Esculetin is the aglycone of esculin and of cichoriin and can obtained by hydrolysis of these molecules. Esculetin has the following chemical formula: ##STR1##
The full chemical name of esculetin is 6,7-dihydroxy-2H-1-benzopyran-2-one (also known as 6,7-dihydroxycoumarin or cichorigenin).
The presence of .beta.-D-glucosidase has long been regarded as an important diagnostic marker in microbial identification. The most commonly used substrate for the detection of this enzyme is the naturally occurring glycoside esculin (6,7-dihydroxycoumarin-6-glucoside or 6-(.beta.-D-glucopyranosyloxy)-7-hydroxy-2H-1-benzopyran-2-one). ##STR2##
Hydrolysis of esculin yields .beta.-D-glucose and esculetin (6,7-dihydroxycoumarin), the latter compound being detected by the formation of a brown/black complex in the presence of iron salts. This test was first applied in the identification of enterococci and has since found wide application in the identification of other genera (Swan, A. J. Clinical Pathology 7 160-163 (1954), Trepeta et al Antonie van Leewenhoek 53 273-277 (1987)). The main disadvantage of this substrate is that, when incorporated into agar, the resulting complex formed spreads throughout the medium (James et al Zbl. Bakt. Hyg. A267 188-193 (1987)). This creates difficulties in distinguishing .beta.-glucosidase producing colonies when present within a mixed culture.
Synthetic substrates are also available for detection of .beta.-D-glucosidase to yield either chromogenic or fluorescent compounds upon hydrolysis (Manafi et al Microbiol. Reviews 55 335-348 (1991)). For example, the fluorogenic compound 4-methylumbelliferyl-.beta.-D-glucoside has been widely used although it has several disadvantages when incorporated into an agar medium. These include the fact that the recognition of colonies can only be performed under the presence of long wave ultraviolet light and also the glucose released from the substrate by hydrolysis, may be utilised by the organisms to produce acid. This causes a reduction in the fluorescence produced by 4-methylumbelliferone due to the predominance of the undissociated form of the molecule at low pH. In addition, the released umbelliferone has a tendency to diffuse through the agar, creating difficulties distinguishing individual colonies producing the target enzyme. Other substrates commonly employed include .beta.-D-glucoside derivatives of nitrophenol (Trepeta et al Antonie van Leewenhoek 53 273-277 (1987)). However, widespread diffusion is again a severely limiting factor when incorporating such substrates into solid media (Manafi et al Microbiol. Reviews 55 335-348 (1991)).
.beta.-galactosidase is also an important diagnostic marker in microbial identification. It is perhaps the most widely studied of all microbial enzymes and its presence has long been recognied as a valuable taxonomic marker. This is particularly true in the bacterial family Enterobacteriaceae where assay of .beta.-galactosidase has been used for many years for the differentiation of non-lactose fermenting species from slow or late lactose fermenters (James, A. L., In Chemical Methods in Prokazyotic Systematics, 471-492, ed. Goodfellow and O'Donnell, Wiley & Sons (1994)). Numerous substrates are available for the detection of .beta.-galactosidase, the most common being ortho-nitrophenyl .beta.-D-galactoside (ONPG) which releases yellow o-nitrophenol upon hydrolysis (Lowe, G. H., J. Medical Laboratory Technology 19 21 (1962)). Fluorogenic substrates have also been used utilising labels such as resorufin, fluorescein and 4-methylumbelliferone (Manafi et al Microbiol. Reviews 55 335-348 (1991), Plovins et al Applied and Environmental Microbiol. 60 4638-4641 (1994)).
An important application of the .beta.-galactosidase assay is the detection of "coliforms" in water and food samples. This has led to the development of membrane filtration techniques which incorporate a suitable substrate for the direct detection of .beta.-galactosidase (Brenner et al Applied and Environmental Microbiol. 59 3534-3544 (1993), Ceneci et al Microbios 76 47-54 (19 )). The most widely used substrate for this purpose is 4-methylumbelliferone .beta.-D-galactoside. For example, this substrate was used in a rapid assay which allowed detection of as few as 1 faecal coliform per 100 ml in six hours (Berg et al Applied and Environmental Microbiol. 54 2118-2122 (1988)). The limitations of this substrate are that the released 4-methylumbelliferone readily diffuses across the filter and the fluorescence produced can only be visualised under ultra-violet light.
Due to the limitations of these substrates for identification of .beta.-glucosidase and .beta.-galactosidase, chromogenic compounds have been employed which yield insoluble products upon hydrolysis. Such substrates provide the advantage that the released chromogen remains localised around the bacterial colony without diffusing through the medium (Kodaka et al J. Clinical Microbiol. 33 199-201 (1995)). Examples of these for the detection of .beta.-glucosidase include, indoxyl .beta.-D-glucoside and 5-bromo-4-chloro-3-indolyl .beta.D-glucoside and examples for the detection of .beta.-galactosidase include galactosides of indoxyl and its halogenated derivatives such as 5-bromo-4-chloro-3-indolyl .beta.-D-galactoside (X-gal) (Kodaka et al J. Clinical Microbiol. 33 199-201 .(1995)). The aglycone released by hydrolysis from these substrates is rapidly oxidised by air to form a purple/blue indigoid dye on the colony mass (James, A. L., In Chemical Methods in Prokaryotic Systematics, 471-492, ed. Goodfellow and O'Donnell, Wiley & Sons (1994)). Whilst these substrates are highly effective, they are relatively difficult to prepare and although commercially available, their extremely high cost has proved prohibitive for large scale diagnostic use. The use of 8-hydroxyquinoline .beta.-D-glucoside has also been described as an alternative to esculin for the detection of .beta.-glucosidase (James et al Zbl. Bakt. Hyg. A267 188-193 (1987)). Although impressive results were obtained, toxicity problems have been encountered particularly with Gram positive organisms (Albert et al British Journal of Experimental Pathology 34 119-130 (1953)).